The characteristic composition of the intestinal microbiota of breast-fed neonates is due to the presence of particular substances in human milk. This chapter aims to identify the components that are able to promote the prebiotic effect in human milk, and is oriented towards modifying the composition of infant formulas in order to obtain an intestinal microbiota similar to that of breast-fed babies. It discusses history, synthesis, structure, metabolism of human milk oligosaccharides (HMO). No natural substances have the same biochemical composition as that of HMO, nor can they be synthesized in large quantities at acceptable prices. To overcome these problems, the industry has focused on the production of several carbohydrates, so-called nondigestible oligosaccharides (NDO), which, although having compositions different from those of HMO, are able to selectively stimulate the growth of bifidobacteria and lactobacilli in the colon, reproducing the prebiotic effects of HMO. In conclusion, data related to the use of NDO in infant formulas clearly show the efficacy of galactooligosaccharides (GOS)/inulin mixtures, the capacity of GOS to induce a bifidogenic effect, even if in only a small number of studies, and the positive effects of FOS at a concentration of 4.0 g/liter on the intestinal microbiota. It has been demonstrated that, due to the presence of resistant bonds in their molecules, NDO are able to exert a prebiotic effect. This further confirms that, because of their peculiar structure, HMO have a very significant role in modulating the intestinal microbiota of neonates.